The Zahn drawings: new illustrations of Xenopus embryo and tadpole stages for studies of craniofacial development.

The embryos and tadpoles of the frog Xenopus are increasingly important subjects for studies of the development of the head and face - studies that are providing novel and crucial insight into the causes and prevention of a suite of devastating birth defects, as well as basic evolutionary and developmental biology. However, many studies are conducted on a range of embryonic stages that are not fully represented in the beloved Xenopus resource, Nieuwkoop and Faber's classic Normal Table of Xenopus laevis (Daudin) The lack of standardized images at these stages acts as a barrier to the efficient and accurate representation and communication of experimental methodology and expression data. To fill this gap, we have created 27 new high-quality illustrations. Like their oft-used predecessors from Nieuwkoop and Faber, these drawings can be freely downloaded and used, and will, we hope, serve as an essential resource for this important model system.

Long-Term, Stochastic Editing of Regenerative Anatomy via Targeting Endogenous Bioelectric Gradients.

We show that regenerating planarians' normal anterior-posterior pattern can be permanently rewritten by a brief perturbation of endogenous bioelectrical networks. Temporary modulation of regenerative bioelectric dynamics in amputated trunk fragments of planaria stochastically results in a constant ratio of regenerates with two heads to regenerates with normal morphology. Remarkably, this is shown to be due not to partial penetrance of treatment, but a profound yet hidden alteration to the animals' patterning circuitry. Subsequent amputations of the morphologically normal regenerates in water result in the same ratio of double-headed to normal morphology, revealing a cryptic phenotype that is not apparent unless the animals are cut. These animals do not differ from wild-type worms in histology, expression of key polarity genes, or neoblast distribution. Instead, the altered regenerative bodyplan is stored in seemingly normal planaria via global patterns of cellular resting potential. This gradient is functionally instructive, and represents a multistable, epigenetic anatomical switch: experimental reversals of bioelectric state reset subsequent regenerative morphology back to wild-type. Hence, bioelectric properties can stably override genome-default target morphology, and provide a tractable control point for investigating cryptic phenotypes and the stochasticity of large-scale epigenetic controls.

Bioelectric signalling via potassium channels: a mechanism for craniofacial dysmorphogenesis in KCNJ2-associated Andersen-Tawil Syndrome.

KEY POINTS: Xenopus laevis craniofacial development is a good system for the study of Andersen-Tawil Syndrome (ATS)-associated craniofacial anomalies (CFAs) because (1) Kcnj2 is expressed in the nascent face; (2) molecular-genetic and biophysical techniques are available for the study of ion-dependent signalling during craniofacial morphogenesis; (3) as in humans, expression of variant Kcnj2 forms in embryos causes a muscle phenotype; and (4) variant forms of Kcnj2 found in human patients, when injected into frog embryos, cause CFAs in the same cell lineages. Forced expression of WT or variant Kcnj2 changes the normal pattern of Vmem (resting potential) regionalization found in the ectoderm of neurulating embryos, and changes the normal pattern of expression of ten different genetic regulators of craniofacial development, including markers of cranial neural crest and of placodes. Expression of other potassium channels and two different light-activated channels, all of which have an effect on Vmem , causes CFAs like those induced by injection of Kcnj2 variants. In contrast, expression of Slc9A (NHE3), an electroneutral ion channel, and of GlyR, an inactive Cl(-) channel, do not cause CFAs, demonstrating that correct craniofacial development depends on a pattern of bioelectric states, not on ion- or channel-specific signalling. Using optogenetics to control both the location and the timing of ion flux in developing embryos, we show that affecting Vmem of the ectoderm and no other cell layers is sufficient to cause CFAs, but only during early neurula stages. Changes in Vmem induced late in neurulation do not affect craniofacial development. We interpret these data as strong evidence, consistent with our hypothesis, that ATS-associated CFAs are caused by the effect of variant Kcnj2 on the Vmem of ectodermal cells of the developing face. We predict that the critical time is early during neurulation, and the critical cells are the ectodermal cranial neural crest and placode lineages. This points to the potential utility of extant, ion flux-modifying drugs as treatments to prevent CFAs associated with channelopathies such as ATS. ABSTRACT: Variants in potassium channel KCNJ2 cause Andersen-Tawil Syndrome (ATS); the induced craniofacial anomalies (CFAs) are entirely unexplained. We show that KCNJ2 is expressed in Xenopus and mouse during the earliest stages of craniofacial development. Misexpression in Xenopus of KCNJ2 carrying ATS-associated mutations causes CFAs in the same structures affected in humans, changes the normal pattern of membrane voltage potential regionalization in the developing face and disrupts expression of important craniofacial patterning genes, revealing the endogenous control of craniofacial patterning by bioelectric cell states. By altering cells' resting potentials using other ion translocators, we show that a change in ectodermal voltage, not tied to a specific protein or ion, is sufficient to cause CFAs. By adapting optogenetics for use in non-neural cells in embryos, we show that developmentally patterned K(+) flux is required for correct regionalization of the resting potentials and for establishment of endogenous early gene expression domains in the anterior ectoderm, and that variants in KCNJ2 disrupt this regionalization, leading to the CFAs seen in ATS patients.

V-ATPase-dependent ectodermal voltage and pH regionalization are required for craniofacial morphogenesis.

Using voltage and pH reporter dyes, we have discovered a never-before-seen regionalization of the Xenopus ectoderm, with cell subpopulations delimited by different membrane voltage and pH. We distinguished three courses of bioelectrical activity. Course I is a wave of hyperpolarization that travels across the gastrula. Course II comprises the appearance of patterns that match shape changes and gene expression domains of the developing face; hyperpolarization marks folding epithelium and both hyperpolarized and depolarized regions overlap domains of head patterning genes. In Course III, localized regions of hyperpolarization form at various positions, expand, and disappear. Inhibiting H(+) -transport by the H(+) -V-ATPase causes abnormalities in: (1) the morphology of craniofacial structures; (2) Course II voltage patterns; and (3) patterns of sox9, pax8, slug, mitf, xfz3, otx2, and pax6. We conclude that this bioelectric signal has a role in development of the face. Thus, it exemplifies an important, under-studied mechanism of developmental regulation.

Bioelectricity: Reasons to Be Amazed and Hopeful-My Personal Favorites.

The field of bioelectricity is growing so rapidly, any attempt on my part to summarize its extraordinary breadth and depth would be a shallow report, a list really. Instead, I've zoomed in to introduce two marvelous examples of new understanding and application of bioelectricity. The signaling Venus flytraps use to capture and digest insects is a marvel of electrical engineering. The application of magnetic fields to treat conditions with brain involvement can offer relief from debilitating symptoms that have been refractory to other treatments. These, and a multitude of other fascinating stories, are helping to spread the word that bioelectricity can and should be seen as a critical approach that can change both science and medicine. This important journal will continue to carry that message.

Preventing Ethanol-Induced Brain and Eye Morphology Defects Using Optogenetics.

Background: Embryonic exposure to the teratogen ethanol leads to dysmorphias, including eye and brain morphology defects associated with fetal alcohol spectrum disorder (FASD). Exposure of Xenopus laevis embryos to ethanol leads to similar developmental defects, including brain and eye dysmorphism, confirming our work and the work of others showing Xenopus as a useful system for studies of the brain and eye birth defects associated with FASD. Several targets of ethanol action have been hypothesized, one being regulation of Kir2.1 potassium channel. Endogenous ion fluxes and membrane voltage variation (bioelectric signals) have been shown to be powerful regulators of embryonic cell behaviors that are required for correct brain and eye morphology. Disruptions to these voltage patterns lead to spatially correlated disruptions in gene expression patterns and corresponding morphology. Materials and Methods: Here, we use controlled membrane voltage modulation to determine when and where voltage modulation is sufficient to rescue ethanol-induced brain and eye defects in Xenopus embryos. Results: We found (1) that modulating membrane voltage using light activation of the channelrhodopsin-2 variant D156A rescues ethanol exposed embryos, resulting in normal brain and eye morphologies; (2) hyperpolarization is required for the full duration of ethanol exposure; (3) hyperpolarization of only superficial ectoderm is sufficient for this effect; and(4) the rescue effect acts at a distance. Conclusions: These results, particularly the last, raise the exciting possibility of using bioelectric modulation to treat ethanol-induced brain and eye birth defects, possibly with extant ion channel drugs already prescribed to pregnant women. This may prove to be a simple and cost-effective strategy for reducing the impact of FASD.

Live imaging of intracellular pH in planarians using the ratiometric fluorescent dye SNARF-5F-AM.

Physiological parameters such as resting potential and pH are increasingly recognized as important regulators of cell activity and tissue-level events in regeneration, development, and cancer. The availability of fluorescent reporter dyes has greatly increased the ability to track these properties in vivo. The planarian flatworm is an important and highly tractable model system for regeneration, stem cell biology, and neuroscience; however, no protocols have been published for investigating pH in this system. Here, we report a simple and effective protocol for imaging pH gradients in living planaria suitable for intact and regenerating flatworms.

Light-activation of the Archaerhodopsin H(+)-pump reverses age-dependent loss of vertebrate regeneration: sparking system-level controls in vivo.

Optogenetics, the regulation of proteins by light, has revolutionized the study of excitable cells, and generated strong interest in the therapeutic potential of this technology for regulating action potentials in neural and muscle cells. However, it is currently unknown whether light-activated channels and pumps will allow control of resting potential in embryonic or regenerating cells in vivo. Abnormalities in ion currents of non-excitable cells are known to play key roles in the etiology of birth defects and cancer. Moreover, changes in transmembrane resting potential initiate Xenopus tadpole tail regeneration, including regrowth of a functioning spinal cord, in tails that have been inhibited by natural inactivity of the endogenous H(+)-V-ATPase pump. However, existing pharmacological and genetic methods allow neither non-invasive control of bioelectric parameters in vivo nor the ability to abrogate signaling at defined time points. Here, we show that light activation of a H(+)-pump can prevent developmental defects and induce regeneration by hyperpolarizing transmembrane potentials. Specifically, light-dependent, Archaerhodopsin-based, H(+)-flux hyperpolarized cells in vivo and thus rescued Xenopus embryos from the craniofacial and patterning abnormalities caused by molecular blockade of endogenous H(+)-flux. Furthermore, light stimulation of Arch for only 2 days after amputation restored regenerative capacity to inhibited tails, inducing cell proliferation, tissue innervation, and upregulation of notch1 and msx1, essential genes in two well-known endogenous regenerative pathways. Electroneutral pH change, induced by expression of the sodium proton exchanger, NHE3, did not rescue regeneration, implicating the hyperpolarizing activity of Archaerhodopsin as the causal factor. The data reveal that hyperpolarization is required only during the first 48 hours post-injury, and that expression in the spinal cord is not necessary for the effect to occur. Our study shows that complex, coordinated sets of stable bioelectric events that alter body patterning-prevention of birth defects and induction of regeneration-can be elicited by the temporal modulation of a single ion current. Furthermore, as optogenetic reagents can be used to achieve that manipulation, the potential for this technology to impact clinical approaches for preventive, therapeutic, and regenerative medicine is extraordinary. We expect this first critical step will lead to an unprecedented expansion of optogenetics in biomedical research and in the probing of novel and fundamental biophysical determinants of growth and form.

Long-distance signals are required for morphogenesis of the regenerating Xenopus tadpole tail, as shown by femtosecond-laser ablation.

BACKGROUND: With the goal of learning to induce regeneration in human beings as a treatment for tissue loss, research is being conducted into the molecular and physiological details of the regeneration process. The tail of Xenopus laevis tadpoles has recently emerged as an important model for these studies; we explored the role of the spinal cord during tadpole tail regeneration. METHODS AND RESULTS: Using ultrafast lasers to ablate cells, and Geometric Morphometrics to quantitatively analyze regenerate morphology, we explored the influence of different cell populations. For at least twenty-four hours after amputation (hpa), laser-induced damage to the dorsal midline affected the morphology of the regenerated tail; damage induced 48 hpa or later did not. Targeting different positions along the anterior-posterior (AP) axis caused different shape changes in the regenerate. Interestingly, damaging two positions affected regenerate morphology in a qualitatively different way than did damaging either position alone. Quantitative comparison of regenerate shapes provided strong evidence against a gradient and for the existence of position-specific morphogenetic information along the entire AP axis. CONCLUSIONS: We infer that there is a conduit of morphology-influencing information that requires a continuous dorsal midline, particularly an undamaged spinal cord. Contrary to expectation, this information is not in a gradient and it is not localized to the regeneration bud. We present a model of morphogenetic information flow from tissue undamaged by amputation and conclude that studies of information coming from far outside the amputation plane and regeneration bud will be critical for understanding regeneration and for translating fundamental understanding into biomedical approaches.

Making solutions from dry chemicals.

INTRODUCTIONSolution making most typically involves dissolving a dry chemical in water or other specified solvent. The amount of chemical to be added to a solvent depends on the final concentration or molarity (M) needed for the finished solution and the total amount in liters (L) of solution required. The easiest way to measure chemicals is by mass. Typically, to make a solution one must determine the mass of chemical needed, based on the desired final concentration (usually molarity), the molecular weight (MW) of the chemical, and the final volume of solution. This article describes the calculations involved in making solutions from dry chemicals.

Making solutions from hydrated compounds.

INTRODUCTIONSolution making typically involves dissolving dry chemicals in water or other specified solvent. The amount of chemical to be added to a solvent depends on the final concentration or molarity (M) needed for the finished solution and the total amount in liters (L) of solution required. However, some chemicals come with water molecules attached. The molecular weight (MW) of such compounds, listed as formula weight (FW) on the bottle, includes the mass of the water. Whenever you would use the MW of an unhydrated compound in calculations, use instead the MW of the hydrated compound. If a recipe tells how many grams to use of the unhydrated compound, determine the target concentration and then calculate the grams to use of hydrated compound. When using a hydrated compound, the attached water molecules contribute water to the solution, potentially diluting the final concentration (if the solvent is water). Therefore, you must account for the contribution of water from the hydrated compound when determining the volume of solvent (water) to add. This article describes the calculations involved in making solutions from hydrated compounds.

Making and diluting stock solutions.

INTRODUCTIONFor particular experiments, certain solutions are used frequently and are therefore made up in large quantities. To minimize the volume actually occupied by these solutions, they are often made at a higher concentration than that which will be used. These concentrated solutions are referred to as stock solutions. Stock solutions save time in addition to space; when you need a solution of a given concentration, you need only dilute the stock rather than starting from scratch. This article describes the steps necessary to make and dilute stock solutions appropriately.

A new tool for tissue engineers: ions as regulators of morphogenesis during development and regeneration.

Currently, most of the research on how to encourage stem cells to replace missing tissues focuses on biochemical control, such as signaling by growth factors. In addition to basic questions, such as how are stem cells induced to differentiate into particular cell types, also inherent in those studies are practical questions about how to identify, grow, induce, and safely deliver stems cells to the proper target. At the Forsyth Center for Regenerative and Developmental Biology, we are examining a different set of signals, specifically bioelectric signals (the regulated movement of ions across membranes), including membrane voltage, pH, and gap junction activity and gating. We have found strong evidence that bioelectrical signals function at many critical, early points, both up- and downstream of transcriptional regulation, during the processes of normal morphogenesis and adult stem cell-based regeneration. Examples described include gap-junction-dependent regulation of stem cell identity in a flatworm, proton-flux-regulated establishment of left-right asymmetry in vertebrates, and proton-flux-initiated regeneration of a complex structure that includes spinal cord--the tadpole tail--in frogs.